1. Field of the Invention
The present invention is directed to improved methods for producing mutant proteins. More specifically, the present invention is directed to methods for producing mutant proteins in vivo utilizing phagemids to isolate mutations away from the chromosome or other DNA of the microorganism in which the DNA has been mutated.
2. State of the Art
A number of methods have been utilized to produce useful mutations in proteins of interest. For example, one method widely used comprises chemical mutagens to treat DNA either in vitro or in vivo. Such methods have commonly utilized chemicals such as nitrosoguanidine, hydroxylamine and/or ultraviolet light to generate the mutations. Such methods, however, can be less desirable because of the uncontrolled nature of the mutations and can often be less than random due to the presence of certain "hot spots" within the DNA. Moreover, only certain types of mutations will result, e.g., transversions or transitions, depending on the chemical used.
Polymerase chain reaction has been used to introduce random mutations in genes which have no selectable phenotype. This in vitro method exploits the inherent infidelity of the Taq DNA polymerase during the reaction whereby varying the conditions can cause the PCR process to produce random mutations. Problems with this technique are caused by the need to reclone the gene of interest into a vector after the reaction and also by the need to conduct individual reactions to guarantee independent mutations due to clonal expansion during amplification. As a result, the method becomes quite time consuming and expensive with a resultant small number of random mutants.
Mutator strains facilitating in vivo mutagenesis are an alternative method to introduce random mutations into a gene. Strains of E. coli that carry mutations in one of the DNA repair pathways have been described which have a higher random mutation rate than that of typical wild type strains (see, e.g., Miller, J., "A Short Course In Bacterial Genetics," Cold Spring Harbor Laboratory Press, 1992, pp.193-211). As reported by Degenen and Cox (J. Bacteriol., 1974, Vol. 117, No. 2, pp.477-487), an E. coli strain carrying a mutD5 allele demonstrates from 100 to 10,000 times the mutation rate of its wild type parent. Greener et al., "Strategies In Molecular Biology," 1994, Vol. 7, pp.32-34, disclosed mutator strain with a mutation rate of one base per 2000 nucleotides. Thus, propagation of a plasmid in a mutator strain will generate mutations in the plasmid and, presumably, in the gene of interest.
Recently, vectors have been developed which combine desirable features of both plasmids and filamentous bacteriophages. As described in Sambrook et al., "Molecular Cloning: A Laboratory Manual," Cold Spring Harbor Press (1989), in their simplest form, these vectors are plasmids with a ColE1 origin of replication and a selectable marker for antibiotic resistance that carry in addition a copy of the major intergenic region of a filamentous bacteriophage. This intergenic region contains all of the sequences required in cis for initiation and termination of viral DNA synthesis and for morphogenesis of bacteriophage particles. Essentially, segments of foreign DNA cloned in these vectors can be propogated as plasmids in the conventional way. When cells harboring these plasmids are infected with a suitable filamentous bacteriophage, the mode of replication of the plasmid changes under the influence of the gene II product coded by the incoming virus. This gene II protein interacts with the intergenic region carried in the plasmid and initiates rolling-circle replication to generate copies of one strand of the plasmid DNA which will be nicked, circularized, and eventually packaged into progeny bacteriophage particles. In the prior art, the single-stranded DNA purified from these particles have been used as a template for sequencing, for oligonucleotide-directed mutagenesis, and for synthesis of strand-specific probes.
Phagemids are desirable cloning vectors for several reasons, most notably that they provide characteristics, such as high stability and yields of double stranded DNA, that are characteristic of conventional plasmids, they circumvent the tedious and time consuming process of subcloning DNA fragments from plasmids to filamentous bacteriophage vectors, and they are sufficiently small that segments of foreign DNA up to 10 kb in length can be obtained in single stranded form.
Despite the knowledge in the art related to mutating DNA and producing resultant valuable mutants, there is a need in the art to easily produce discrete mutations which effect only effect a desired gene of interest, without effecting the host organism.